Lentiviral vectors incorporating linker and tag systems for car detection

Abstract

The invention relates to compositions and methods for cell therapy. Provided are lentiviral vectors comprising, in operable linkage, sequences encoding a Chimeric Antigen Receptor (CAR), a peptide linker, and a tag suitable for detection, selection, or depletion. The CAR comprises an extracellular antigen binding domain, a transmembrane domain, and intracellular signaling domains. The tag, positioned C-terminally or N-terminally to the linker, facilitates detection and / or selection of host cells expressing the CAR. In certain embodiments, the tag comprises a truncated, non-functional cell surface protein (e.g., hEGFRt) usable as a safety switch for in vivo depletion via cognate binding agents (e.g., antibodies). Also provided are genetically engineered host cells (e.g., T cells, NK cells) transduced with said vectors, pharmaceutical compositions comprising such cells, methods for their manufacture, and methods for treating diseases, such as cancer, using said compositions.

Description

LENTIVIRAL VECTORS INCORPORATING LINKER AND TAG SYSTEMS FOR CAR DETECTIONRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63,641,846 filed May 02, 2024, which is hereby incorporated by reference in its entirety. All documents cited or referenced herein (“herein cited documents”), together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.FIELD OF THE INVENTION

[0002] This invention is related to novel compositions and methods related to cell therapy development and manufacturing.BACKGROUND OF THE INVENTION

[0003] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

[0004] Cell therapies, particularly Chimeric Antigen Receptor T-Cells (CAR-T) and therapies derived from Induced Pluripotent Stem Cells (iPSCs), have shown great promise in treating various diseases, especially in the field of cancer immunotherapy. Two prominent cell therapy modalities are Chimeric Antigen Receptor T-Cells (CAR-T) and therapies derived from Induced Pluripotent Stem Cells (iPSCs). CAR-T cells are genetically engineered to express a synthetic receptor that targets specific antigens on cancer cells, enabling them to recognize and eliminate tumor cells. iPSCs, on the other hand, are derived from adult somatic cells that have been reprogrammed to a pluripotent state, allowing them to differentiate into various cell types, including immune cells such as natural killer (NK) cells.SUMMARY OF THE INVENTION

[0005] In one aspect, this disclosure relates to improved methods and systems for the efficient manufacture of CAR-T and iPSC-derived cell therapies while maintaining high quality and safety standards. Despite the remarkable potential of CAR-T and iPSC-derived cell therapies, their widespread adoption has been hindered by the complex, time-consuming, and expensive manufacturing processes. Current manufacturing methods often involve multiple manual steps, lack of standardization, and require specialized equipment and skilled personnel. These challenges result in limited scalability, high production costs, and reduced accessibility to patients in need. To address these limitations, the instant disclosure features improved methods and systems for efficiently manufacturing CAR-T and iPSC-derived cell therapies while maintaining high quality and safety standards. In one aspect, the improved methods and systems featured herein provide a flexible, modular, and customizable manufacturing platform that adapts to industry innovations and streamlines the production process for these life-saving therapies. Specifically, in one aspect, the disclosure relates to novel lentiviral vector constructs incorporating integrated linkers and tags (e.g., Tag / GFP or truncated receptors) that enhance CAR detection, enable standardized manufacturing workflows, and in some aspects, provide safety mechanisms for clinical use. In an aspect, this disclosure relates to a lentiviral vector comprising, in operable linkage: a 5' long terminal repeat (LTR); a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) protein, wherein the CAR protein comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain; a nucleic acid sequence encoding a first peptide linker; a nucleic acid sequence encoding a tag, wherein the tag is suitable for detection, selection, or depletion of a host cell expressing the CAR protein; and a 3' long terminal repeat (LTR).

[0006] In certain aspects, the disclosure provides a lentiviral vector comprising, in operable linkage from 5' to 3': a 5' long terminal repeat (LTR); a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) protein, wherein the CAR protein comprises an extracellular antigen binding domain (which in some embodiments comprises an internal, second peptide linker, e.g., within an scFv), a transmembrane domain, and an intracellular signaling domain comprising at least a primary activation domain and at least one co-stimulatory domain;a nucleic acid sequence encoding a first peptide linker; a nucleic acid sequence encoding a tag suitable for detection, selection, or depletion of a host cell expressing the CAR protein; and a 3' long terminal repeat (LTR).

[0007] In some aspects, the extracellular antigen binding domain comprises a single-chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL). In some aspects, the extracellular antigen binding domain may comprise other molecular structures capable of specific antigen binding, including but not limited to, antigen-binding fragments such as Fab fragments, single-domain antibodies (e.g., VHH fragments or nanobodies), receptor ectodomains or ligands that bind a target receptor, designed ankyrin repeat proteins (DARPins), or other protein scaffolds engineered or selected for binding to the desired target antigen. In some aspects where the extracellular antigen binding domain comprises an scFv, a second peptide linker connects the VH and VL regions. In certain aspects, this second peptide linker is a Gly4Ser linker comprising the sequence (GlyGlyGlyGlySer)n, wherein n is 1, 2, 3, 4, or 5.

[0008] In some aspects, the extracellular antigen binding domain specifically binds an antigen selected from the group consisting of BCMA, CD19, CD20, CD22, CD30, CD123, CEA, CLL-1, EGFRvIII, FAP, FLT3, GD2, GPC3, HER2, HERV-K env, Mesothelin, MUC1, PSMA, ROR1, TAG72, Viral antigens (e.g., from CMV, EBV, HIV), or any other suitable target antigen featured herein.

[0009] In certain aspects, the nucleic acid sequence encoding the tagis positioned 3' to the nucleic acid sequence encoding the first peptide linker, such that the tag is expressed as part of a CAR-linker-tag fusion protein. In certain embodiments, the tag comprises a truncated human cell surface receptor or an antibody-binding fragment thereof, wherein said truncated receptor or fragment lacks native signaling function.

[0010] In certain embodiments, the tag comprises a truncated human Epidermal Growth Factor Receptor (hEGFRt). In some embodiments, this truncated hEGFRt is detectable by an anti-EGFR antibody selected from cetuximab, panitumumab, or any other anti-EGFR antibody featured herein. In certain embodiments, the tag comprises a truncated CD34, truncated CD19, truncated CD20, truncated low-affinity nerve growth factor receptor (LNGFR), or other suitable truncated receptors featured herein.

[0011] In some aspects, the tag comprises a fluorescent protein. In some embodiments, the fluorescent protein is Green Fluorescent Protein (GFP) or mNeon Green.

[0012] In some aspects, the tag is suitable for detection by flow cytometry using an antibody or reagent that binds the tag. In certain aspects, the tag serves as a selection marker for enriching host cells expressing the CAR-linker-tag fusion protein. In certain embodiments, the tag serves as a safety switch enabling depletion of host cells expressing the CAR-linker-tag fusion protein upon administration of an agent that binds the tag. In certain aspects, a tag comprising truncated hEGFRt is configured to function as a safety switch enabling selective depletion of cells expressing the CAR-linker-tag fusion protein upon administration of an anti- EGFR antibody, such as cetuximab or panitumumab. In some aspects the same tag is capable of use as a detection tag, a selection tag and / or a depletion tag, when used in different methods of this disclosure. In some aspects, the intracellular signaling domain comprises a primary activation domain derived from CD3^ other CD3 chains (CD3y, CD35, CD3s), the Fc receptor gamma chain (FcsRIy), or functional variants or fragments thereof.

[0013] In some aspects, the intracellular signaling domain further comprises at least one co-stimulatory domain derived from a protein selected from the group consisting of CD28, 4- 1BB (CD137), ICOS, 0X40 (CD134), CD27, CD2, DAP10, GITR, CD30, CD40, PD-1 (in inhibitory contexts or modified forms), LFA-1, CD7, NKG2C, or any other suitable costimulatory domain featured herein.

[0014] In an aspect, the disclosure relates to a genetically engineered host cell transduced with the lentiviral vector described herein, wherein the lentiviral vector is integrated into the genome of the host cell, and wherein the host cell expresses the CAR-linker-tag fusion protein.

[0015] . In some aspects, the host cell is a human T cell. In some embodiments, the T cell is selected from the group consisting of a pan T-cell, a CD4+ T cell, a CD8+ T cell, a gammadelta (y5) T-cell, a regulatory T-cell (Treg), a memory T cell (including central memory (TCM), effector memory (TEM), stem cell memory (TSCM), or tissue-resident memory T cells (TRM)), a naive T cell (TN), a tumor-infiltrating lymphocyte (TIL), or any other T cell subset featured herein. In some embodiments, the host cell is a Natural Killer (NK) cell, an NKT cell, a macrophage, a dendritic cell, an Induced Pluripotent Stem Cell (iPSC)-derived cell (e.g., iPSC-derived T cell, NK cell, or macrophage), an embryonic stem cell (ESC)-derived cell, or a hematopoietic stem / progenitor cell (HSPC).

[0016] In certain aspects, the disclosure provides a method for manufacturing genetically engineered host cells expressing a CAR-linker-tag fusion protein, the method comprising: obtaining a population of host cells from a subject or donor; activating the host cells ex vivo',transducing the activated host cells with any lentiviral vector featured herein wherein the lentiviral vector comprises, in operable linkage from 5' to 3', a 5' LTR, a nucleic acid sequence encoding a CAR protein comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, a nucleic acid sequence encoding a first peptide linker, a nucleic acid sequence encoding a tag suitable for detection, selection, or depletion of the host cells, and a 3' LTR, thereby generating CAR-transduced host cells comprising the lentiviral vector integrated into their genome and expressing the CAR- linker-tag fusion protein; detecting and / or selecting the CAR-transduced host cells based on expression of the tag; expanding the detected and / or selected CAR-transduced host cells ex vivo in a bioreactor system; and monitoring the expanded CAR-transduced host cells for CAR-linker-tag fusion protein expression using detection reagents specific for the tag.

[0017] In certain aspects, the disclosure relates to a method for manufacturing chimeric antigen receptor T-cells (CAR-T cells), comprising: isolating T-cells from a patient or donor; activating the T-cells ex vivo', transducing the activated T-cells with any lentiviral vector featured herein, e.g., in some aspects a lentiviral vector comprising nucleic acid sequences encoding a CAR protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, a first peptide linker, and a tag suitable for detection or selection of transduced T-cells; expanding the transduced T-cells ex vivo in a bioreactor system to generate a population of CAR-T cells expressing the CAR-linker-tag fusion protein; and harvesting the CAR-T cells.

[0018] In some aspects, the methods further comprise the step of detecting the expression of the CAR-linker-tag fusion protein or the tag on the surface of the transduced host cells or T- cells using flow cytometry. In some embodiments, detecting comprises contacting the cells with an antibody or reagent that specifically binds to the tag. In other embodiments, the methods further comprise enriching the population of transduced host cells or T-cells based on the expression of the tag. In certain aspects, the methods further comprise harvesting, washing, and formulating the expanded transduced host cells or T-cells into a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier. In some embodiments, the method further comprises cryopreserving the pharmaceutical composition.

[0019] In another aspect, the disclosure provides a pharmaceutical composition comprising a population of the genetically engineered host cells described herein and a pharmaceutically acceptable carrier.

[0020] In yet another aspect, the disclosure relates to a method for controlling persistence or activity of CAR-expressing cells in a subject, the method comprising:(1) administering to the subject a population of cells expressing a CAR- linker-tag fusion protein encoded by any lentiviral vector featured herein, wherein the tag comprises a moiety targetable for depletion (e.g., a truncated human cell surface receptor or an antibodybinding fragment thereof, where the truncated receptor or fragment lacks native signaling function, such as a truncated hEGFRt) ; and(2) subsequently administering an agent that binds the tag (e.g., anti-tag antibody, such as an anti-EGFR antibody like cetuximab or panitumumab) to the subject in an amount effective to deplete at least a portion of the CAR-expressing cells when reduction of CAR-T cell activity is clinically indicated.

[0021] In an aspect, the disclosure provides a method for treating a cancer in a subject, the cancer characterized by cells expressing a target antigen, the method comprising administering to the subject a therapeutically effective amount of the genetically engineered host cells described herein, wherein the extracellular antigen binding domain of the CAR protein specifically binds the target antigen.

[0022] In an aspect, the disclosure relates to a method for manufacturing allogeneic cell therapy products, the method comprising: genetically modifying induced pluripotent stem cells (iPSCs) with any lentiviral vector as described herein to generate CAR-expressing iPSCs capable of expressing a CAR-linker- tag fusion protein; differentiating the CAR-expressing iPSCs into immune effector cells; and expanding the differentiated immune effector cells to generate a population of allogeneic CAR-expressing immune effector cells.

[0023] In certain embodiments, the immune effector cells are natural killer (NK) cells, T cells, or macrophages, or any other immune effector cell featured herein. In some aspects, the differentiation comprises: differentiating the CAR-expressing iPSCs into hematopoietic progenitor cells (HPCs); and further differentiating the HPCs into the immune effector cells.

[0024] In an aspect, the disclosure provides a pharmaceutical composition comprising allogeneic CAR-expressing immune effector cells produced by the methods described herein and a pharmaceutically acceptable carrier.

[0025] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0026] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0027] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0029] Fig. 1: Overview of the UpTempoSMCAR-T platform. Fig. 1 provides a schematic overview of the UpTempoSMCAR-T platform, a flexible, modular, and customizableplatform process for manufacturing autologous CAR-T cell therapies. The platform consists of several modules, including cell thawing and culture, wash and transduction, cell expansion, and harvest, fill, and finish. The Fig. illustrates the key components and devices used in each module, such as the Plasmatherm for cell thawing, CliniMACS Prodigy for cell selection, Sepax for cell washing and transduction, Quantum and G-Rex devices for cell expansion, and Sepax C-Pro and Finia for harvest, fill, and finish. The arrows indicate the sequential flow of the manufacturing process from one module to the next.

[0030] Figs. 2A-2C: Thawing culture module. Fig. 2A provides a detailed illustration of the thawing culture module in the UpTempoSMCAR-T platform. The module involves the thawing of patient-derived T-cells using the Plasmatherm device, followed by dilution with a thawing solution. The thawed cells are then transferred to a cell culture bag or G-Rex device for initial culture. The Fig. also shows the selection of CD4+ and CD8+ T-cells using the CliniMACS Prodigy device and the activation of the selected T-cells with an activation solution containing anti-CD3 / CD28 antibodies and IL-2. The key steps in the module are numbered from 1 to 4, and the duration of each step is provided. In-process control samples are taken preselection and post-selection (Fig. 2B) for various quality control tests, such as total cell count, cell viability and recovery (Fig. 2C), sterility, and flow cytometry analysis. This process was tested for the harvesting of CD3+ cells. In the depicted example, the viability of total CD3+ cells increased from 91% to 95% post- selection with a 71% recovery.

[0031] Fig. 3A: Wash and transduction module. Fig. 3A illustrates the wash and transduction module in the UpTempoSMCAR-T platform. The activated T-cells from the thawing culture module are washed using the Sepax device to remove any residual activation solution or debris. The washed T-cells are then transduced with a lentiviral vector expressing the CAR-linker-tag fusion protein in a G-Rex CS device. The figure shows the addition of the lentiviral vector and a transduction enhancer (TDX booster) to the T-cells. The transduced T- cells are incubated in the G-Rex CS device for 45-60 minutes. In-process control samples are taken pre-wash and post-wash for quality control tests, such as total cell count, viability, and characterization by flow cytometry for activation, exhaustion, and memory markers.

[0032] Fig. 3B illustrates a schematic representation of the lentiviral vector construct, highlighting the CAR, first linker, and tag (e.g., Tag / GFP) components positioned sequentially between the 5' and 3' LTRs. In the depicted example in Fig. 3A, the CD3+ cells’ viability increased from 83% to 88%, and recovery was 121% pre-wash, and 101% post-wash.

[0033] Figs. 4A-4C: Transduction. Fig. 4A shows anti-CD19 CAR GFP cell counts measured by automated flow cytometry at different MOI values (0, 1, 5, 10, 25, and 50). Fig. 4B displays a graph of anti-CD19 CAR GFP expression (indicating expression of the CAR- linker-GFP fusion protein) in cells transduced in the presence of transducing enhancers at different multiplicity of infection (MOI). Fig. 4C depicts a schematic representation of an example embodiment of a lentiviral vector (LVV) construct design incorporating the CAR, first linker, and tag (e.g., Tag / GFP) components positioned sequentially between the LTRs for detection of the expressed fusion protein.

[0034] Figs. 5A-5C: Cell expansion module. Fig. 5A depicts the cell expansion module in the UpTempoSMCAR-T platform. The transduced T-cells from the wash and transduction module are expanded in a bioreactor system, such as the Quantum or G-Rex devices, to achieve the desired cell numbers. The Fig. shows the seeding of the transduced T-cells into the bioreactor system and the expansion process over a variable length of time, typically 7-10 days. In-process control samples are taken pre-wash, post-wash, and during the expansion phase for quality control tests, such as total cell count, viability, cell characterization by flow cytometry, and metabolite analysis (glucose, lactate, glutamine, ammonia, and lactate dehydrogenase). During this process, the cell health and expansion parameters are measured, these include the cell viability and recovery (Fig. 5B). Fig. 5C shows cell viability and fold increase comparisons between the Quantum and G-Rex devices over a 7-day expansion period. Both devices maintained good viability (>90%), with the Quantum device achieving approximately an 80- fold increase in cell numbers by day 7, compared to approximately a 40-fold increase with the G-Rex device in this example.

[0035] Figs. 6A-6B: Comparison of cell health and expansion using different bioreactor systems. Fig. 6A depicts a comparison of cell health and expansion parameters using different bioreactor systems, such as, for example, the Quantum and G-Rex devices. Both the Quantum and G-Rex devices supported good cell viability (>90%) throughout the expansion period, and the Quantum achieved a significantly higher fold increase in cell numbers compared to the G-Rex device by day 7 in this example. Fig. 6A displays the cell viability and fold increase between the Quantum and G-Rex devices. Both the Quantum and G-Rex devices supported good cell viability (>90%) throughout the expansion period, and the Quantum achieved a significantly higher fold increase in cell numbers compared to the G-Rex device. Fig. 6B illustrates the glucose consumption and lactate production rates observed in the Quantum system during an exemplary expansion run. The data show, for instance, thatglucose consumption rates reached approximately 30 mmol / day by day 4 under these conditions, while lactate production peaked around day 4-5, demonstrating the metabolic activity accompanying cell expansion. Lactate was produced at 25 mmol per day by day 4, and 30 mmol per day by day 7, and similarly glucose was consumed at 30 mmol per day by day 4, and approximately 25 mmol / day by day 7. Lactate concentration was kept below 10 mM using a feeding strategy (meanwhile lactate in G-Rex was higher than 13 mM after day 9 - not shown).

[0036] Figs. 7A-7B: Cell expansion and health using the Xuri vs G-Rex devices. Fig. 7A displays lactate and glucose measurements during cell expansion in the Xuri system. Lactate was produced at 4 mmol per day by day 4, and 17 mmol per day by day 7, while glucose was consumed at 7.5 mmol per day by day 4, and approximately 6 mmol / day by day 7, indicating that the increase of glucose consumption and lactate production is aligned with cell expansion. Fig. 7B displays the cell viability and fold increase between the Xuri and G-Rex devices over a 7-day period. Both devices supported good viability (>90% over the expansion period) with similar fold expansion of cells in this example.

[0037] Figs. 8A-8D: Harvest, fill, and finish module. Fig. 8A illustrates the harvest, fill, and finish module in the UpTempoSMCAR-T platform. The expanded CAR-T cells from the cell expansion module are harvested using the Sepax C-Pro device. The harvested cells are then washed to remove any residual culture media or debris. The washed cells are formulated and filled into the final product containers using the Finia device. The filled product is cryopreserved using a controlled-rate freezer. In-process control samples are taken pre-harvest for quality control tests, such as total cell count, viability, mycoplasma testing, and cell characterization by flow cytometry for CAR-linker-tag fusion protein expression and memory markers. Fig. 8B displays a comparison of exemplary cell viability and recovery percentages obtained using Quantum and G-Rex devices at different stages of the harvest process (preharvest, post-harvested, post- washed). The results indicate high viability (>80-90%) maintained through the harvest and wash steps in both systems under the tested conditions, including essential quality control tests such as total cell count, viability, sterility, and CAR- linker-tag fusion protein expression. Fig. 8C displays the release and characterization parameters available for the final product. Fig. 8D displays the Total Viable Cells (TVC) for the end culture versus the harvested cells on the Quantum and G-Rex devices.

[0038] Fig. 9: Overview of iPSC-based cell therapy solutions. Fig. 9 provides an overview of the applicants’ iPSC-based cell therapy solutions. The figure illustrates the keysteps involved in the production of iPSC-derived cell therapies, starting from the generation of research and GMP iPSC banks. The iPSC banks are be generated, in some aspects, from donor cells through custom reprogramming or obtained from Catalent's off-the-shelf, qualified iPSC banks. The iPSCs then undergo gene engineering to express a desired CAR (e.g., as a CAR- linker-tag fusion protein), followed by differentiation into the target cell type, such as iHSPCs or iNK cells. The differentiated cells are further processed and formulated into the final drug product. The figure highlights the advantages of the applicants’ iPSC banks, including their GMP compliance, commercial exploitability, and availability for licensing.

[0039] Fig. 10: Robust killing by iNK Cells 35 days Post Differentiation. Fig. 10 illustrates the cytotoxic activity of iPSC-derived NK (iNK) cells against K562 target cells. The figure demonstrates cell yield under steady-state conditions from approximately day 35 of differentiation, showing peak cell numbers around 4 million cells per 12-well. The killing assay data shows dose-dependent cytotoxicity with increasing effector-to-target cell ratios (NK:K562), displaying both Caspase 3 / 7 activation and 7-AAD staining as indicators of target cell death. The right panel illustrates the detection principle of the killing assay, including PKH67-labelled target cells, NK effector cells, and the 7-AAD labeling of dead target cells at different effector ratios.

[0040] Fig. 11: Culture Media Optimization to Improve on NK Cell Expansion and Killing Efficacy. Fig. 11 presents a comparison of two different culture media formulations for iNK cell cultivation. The figure displays flow cytometry plots showing CD56bright expression in Medium 1 versus CD56dim expression in Medium 2. The upper graphs demonstrate killing efficacy, with iNK cells in Medium 1 showing substantially higher cytotoxicity against K562 targets compared to those in Medium 2. The lower graphs illustrate proliferation rates, with Medium 2 supporting superior cell expansion over time (up to 9 million cells per 12-well by day 43) compared to Medium 1. The figure also shows NKp44 activation marker expression profiles for cells cultured in different media conditions, demonstrating activation status differences between the media formulations and the effect of adding active components from Medium 1 to cells cultured in Medium 2.DETAILED DESCRIPTION OF THE INVENTION

[0041] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0042] The present disclosure relates to compositions and methods for cellular immunotherapy. Furthermore, the disclosure provides engineered immune cells, such as, e.g., T cells, NK cells, or iPSC-derived cells genetically modified to express a chimeric antigen receptor (CAR) as part of a CAR- linker-tag fusion protein, thereby redirecting their specificity towards target antigens, such as those expressed on cancer cells. The disclosure encompasses the CAR- linker- tag fusion protein constructs themselves, nucleic acids encoding such CAR linker-tag fusion proteins (hereinafter “CAR constructs”), vectors (e.g., viral or non- viral vectors) comprising such nucleic acids, genetically engineered host cells (such as T cells, NK cells, or iPSC-derived cells, hereinafter often referred to as “CAR-expressing cells” or specifically “CAR-T cells” when the host cell is a T cell) comprising such CAR constructs and expressing the -linker-tag fusion protein, methods for producing these engineered host cells, pharmaceutical compositions comprising these engineered host cells, and methods for using these engineered host cells and compositions in immunotherapy to treat or prevent diseases, including but not limited to cancers, infectious diseases, and autoimmune disorders.

[0043] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0044] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” means, in some embodiments, a range of up to ±20%, up to ±10%, up to ±5%, or up to ±1% of a given value.

[0045] The term “antibody” as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. An antibody is, in some embodiments, be a full-length antibody or an antigen-binding fragment thereof.

[0046] The term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, linear antibodies, single domain antibodies (e.g., VHH), and multispecificantibodies formed from antibody fragments, antigen-binding fragments such as Fab fragments, single-domain antibodies (e.g., VHH fragments or nanobodies), receptor ectodomains or ligands that bind a target receptor, designed ankyrin repeat proteins (DARPins), or other protein scaffolds engineered or selected for binding to the desired target antigen

[0047] The term “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected, in some embodiments, with a short linker peptide of ten to about 25 amino acids (referred to herein as a “second linker” or “internal linker” when present within the CAR's antigen-binding domain). The second linker linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, in some embodiments.

[0048] The term “Chimeric Antigen Receptor” or “CAR” as used herein refers to an engineered transmembrane receptor protein, typically expressed on the surface of an immune cell (such as a T cell, NK cell, NKT cell, macrophage, or dendritic cell). In the embodiments described herein, the full expressed construct is typically a fusion protein comprising the CAR protein, a first peptide linker, and a tag. The CAR protein portion itself comprises an extracellular domain capable of binding a specific target antigen, a transmembrane domain, and an intracellular signaling domain capable of initiating an immune response upon antigen binding. In some embodiments, the extracellular antigen binding domain itself comprises an internal (second) peptide linker (e.g., within an scFv).

[0049] The terms “CAR construct” or “nucleic acid encoding a CAR construct” as used herein refer to a nucleic acid sequence (DNA or RNA) that encodes the CAR-linker-tag fusion protein as described herein. This nucleic acid sequence is typically part of an expression cassette contained within a vector. In some aspect, a single CAR construct within a vector comprises sequences encoding multiple distinct CAR proteins or a single CAR protein with multiple distinct extracellular antigen binding domains (e.g., a bi-specific CAR).

[0050] The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, plasmids, cosmids, viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors), and artificial chromosomes. In some embodiments, a single vector encodes all desired genetic elements, including potentially multiple CAR specificities. In some embodiments, multiple vectors are co-introduced into the host cell, each carrying different genetic elements (e.g., different CAR constructs or supplementary molecules).

[0051] The term “lentiviral vector” or “LVV” refers to a vector derived from a lentivirus (a subgroup of retroviruses, e.g., HIV-1) engineered for gene delivery. In some embodiments, lentiviral vectorscan transduce both dividing and non-dividing cells and typically integrate into the host genome.

[0052] The term “long terminal repeat” or “LTR” refers to characteristic sequences found at the ends of retroviral genomes (including lentiviruses), involved in integration and regulation of gene expression. In some embodiments, lentiviral vectors typically contain 5' and 3' LTRs, which are modified (e.g., in SIN vectors).

[0053] The terms “engineered host cell,” or “CAR-expressing cell,” or specifically “CAR- T cell” as used herein refer to any suitable host cell featured herein, including but not limited to a dendritic cell, embryonic stem cell (ESC)-derived cell, hematopoietic stem / progenitor cell (HSPC), Induced Pluripotent Stem Cell (iPSC)-derived cell, macrophage, Natural Killer (NK) cell, NKT cell, or T cell, that has been genetically modified to express a CAR-linker-tag fusion protein.

[0054] The term “first peptide linker” or “linker” as used herein refers primarily to the peptide linker positioned between the CAR coding sequence and the tag coding sequence in the fusion protein construct. This is distinct from a potential “second linker” or “internal linker” that may be present within the CAR's extracellular domain, for example, connecting the variable heavy (VH) and variable light (VL) chains within an scFv. Unless otherwise specified, "linker" refers to the first linker between the CAR and tag.

[0055] The term “tag” as used herein refers to a detectable or selectable moiety, typically a peptide sequence or protein (such as a fluorescent protein or a truncated cell surface receptor), encoded by the CAR construct. In the embodiments described herein, the nucleic acid sequence encoding the tag is positioned ‘to the nucleic acid sequence encoding the first peptide linker, resulting in the tag being expressed as part of a fusion protein at the C-terminus of the first peptide linker. The tag facilitates detection, selection, purification, or provides a safety mechanism for the engineered host cell. Examples of tags include, but are not limited to, fluorescent proteins (e.g., GFP, mNeon Green, mCherry), truncated cell surface receptors (e.g., truncated human EGFR (hEGFRt), CD34, CD19, CD20, LNGFR), or epitope tags (e.g., His- tag, Myc-tag, HA-tag, FLAG-tag).

[0056] The term “extracellular antigen binding domain” refers to the portion of the CAR protein that is exposed extracellularly and is responsible for binding to the target antigen. Examples include antibody fragments, scFvs, nanobodies, or receptor fragments.

[0057] The term “transmembrane domain” refers to the portion of the CAR protein, typically a hydrophobic alpha-helix, that spans the cell membrane and anchors the CAR protein (receptor) in the membrane. Examples include domains derived from CD8a, CD4, CD28, or the CD3(^ chain.

[0058] The term “intracellular signaling domain” refers to the portion of the CAR protein located inside the cell that transmits signals upon binding of the extracellular antigen binding domain to a specific target molecule (e.g., a target antigen). It typically includes a primary activation domain (e.g., derived from CD3Q and at least one or more co- stimulatory domain (e.g., derived from CD28, 4- IBB, 0X40, ICOS).

[0059] The term “co-stimulatory domain” refers to a portion of the CAR intracellular signaling domain derived from a co-stimulatory molecule (e.g., CD28, 4- IBB) that provides signals enhancing T cell activation, proliferation, survival, or persistence.

[0060] The term “activating T cells” refers to the process of stimulating T cells to induce proliferation and render them receptive to gene transfer. Activation occurs, in some embodiments, ex vivo as part of a manufacturing process, or occurs, in other contexts, in vivo. Common methods involve stimulation via CD3 and CD28 pathways. In some aspects, for the ex vivo manufacturing methods described herein, activation is typically performed using methods such as antibody-coated beads (e.g., anti-CD3 / CD28 beads), soluble antibody complexes or nanomatrices, plate-bound antibodies, or artificial antigen-presenting cells (aAPCs) expressing relevant ligands, often in the presence of supporting cytokines (e.g., IL-2, IL-7, IL- 15). In some aspects, examples of, in vivo activation approaches, include, but are not limited to, systemic or targeted administration of activating antibodies (e.g., anti-CD3, anti- CD28), cytokines (e.g., IL-2, IL-15), co-stimulatory receptor agonists, or therapeutic vaccine strategies designed to stimulate T cells within the patient.

[0061] The term “transducing” refers to the process of introducing genetic material (e.g., a CAR construct) into a host cell using a viral vector. “Transfecting” refers to introducing nucleic acids using non-viral methods (e.g., electroporation).

[0062] The term “multiplicity of infection” or “MOI” refers to the ratio of infectious viral particles (e.g., lentiviral vectors) to the number of target cells during transduction.

[0063] The term “bioreactor system” refers to any vessel or system used for the controlled ex vivo culture and expansion of cells, including static culture devices (e.g., flasks, bags, G- Rex®) and dynamic systems (e.g., WAVE™, Quantum®, stirred tanks, Xuri™).

[0064] The terms “treat,” “treating,” and “treatment” as used herein refer to therapeutic measures that cure, slow down, lessen or ameliorate symptoms of, or halt progression of a diagnosed pathologic condition or disorder. As non-limiting examples, the terms “treat,” “treating,” and “treatment” is used in some embodiments to refer to reduction in tumor burden, or improvement in survival or progression free survival.

[0065] The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent (e.g., CAR-expressing cells) that is sufficient to show a benefit to the subject, such as amelioration of symptoms, halting progression, reduction in tumor burden, or improvement in survival.

[0066] The term “subject” or “patient” as used herein refers to a mammal, preferably a human, in need of treatment for a disease or condition.

[0067] The term “pharmaceutically acceptable carrier” as used herein refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound (e.g., engineered cells). Examples include sterile, physiologically compatible buffers or cryopreservation solutions (e.g., PlasmaLyte A, CryoStor® CS5 / CS10) potentially containing excipients like human serum albumin (HSA) or dextran.

[0068] The present disclosure relates to, at least in part, the development of improved steps, methods, and systems for the efficient manufacture of Chimeric Antigen Receptor T-Cells (CAR-T) and Induced Pluripotent Stem Cell (iPSC)-derived cell therapies while maintaining high quality and safety standards. The methods, constructs, and systems described herein address the challenges associated with conventional manufacturing processes, which are often complex, time-consuming, variable, and expensive. By providing innovations such as integrated detection systems within CAR constructs (e.g., the first linker followed by a tag described herein) and flexible, modular manufacturing platforms, the present disclosure streamlines the production process. The improved processes, constructs, and systems described herein may lead, at least in part, to one or more of the following advantageous outcomes:1. Facilitated detection and characterization: Integration of a specific first peptide linker followed by a tag (e.g., a truncated receptor, e.g., as a non-limiting example, hEGFRt or a fluorescent protein, e.g., as a non-limiting example, GFP) C-terminal to the CAR protein allows for, in some embodiments, reliable, specific, and / or standardized detection, quantification, and characterization of CAR- linker- tag fusion protein expression in engineered host cells using readily available reagents (e.g., as non-limiting examples, the use ofcommercial antibodies via flow cytometry or direct fluorescence detection), simplifying process monitoring (e.g., transduction efficiency assessment), and final product release testing.2. Improved manufacturing consistency and efficiency: Standardized detection methods enabled by the incorporated linker-tag system, combined with a modular manufacturing platform (like the described UpTempoSMplatform), in some embodiments provide improved process consistency, robustness, reproducibility, and potentially reduced processing times or costs compared to methods relying on bespoke detection reagents or less integrated workflows.3. Enhanced process control: The incorporated tag, is used, in some embodiments not only for detection but also for selection and / or enrichment of CAR-expressing cells during manufacturing (e.g., as non-limiting examples, by using FACS or magnetic beads targeting the tag), potentially leading to a final cell product with higher purity and more defined characteristics.4. Potential for in vivo tracking and safety: Certain tags as featured herein (e.g., as a non-limiting example, truncated hEGFRt) allow for potential in vivo tracking of the administered CAR-T cells and serve, in some embodiments, as a safety switch, enabling depletion of the cells through administration of a corresponding antibody (e.g., cetuximab) if needed.5. Streamlined CAR development and comparison: The vectors are designed, in some embodiments, to permit the CAR-encoding sequences to be swapped while retaining the first linker and tag components. The use of the novel vector design featured herein, and these exemplary vectors facilitates more rapid comparison and characterization (e.g., binding, efficacy) of different CAR protein candidates during preclinical development.6. Platform flexibility and adaptability: The described modular manufacturing platform, integrated with the novel vector design, allows adaptation to different CAR targets, cell types (including T cells and potentially iPSC-derived cells), and evolving manufacturing technologies, supporting broader application.7. Support for high-quality cell therapy production: In some embodiments, the overall system produces CAR-T and other engineered cell therapies efficiently while maintaining high quality and safety standards suitable for clinical application.

[0069] In some embodiments, the methods and systems of this disclosure, as a non-limiting example, provide a vector system, e.g., lentiviral vector system, for the efficient transduction of host cells (e.g., T-cells) to express a chimeric antigen receptor (CAR) fusion proteincomprising the CAR, a first peptide linker, and a tag. The lentiviral vector comprises, in operable linkage from 5' to 3', a 5' long terminal repeat (LTR), a sequence encoding the CAR- linker-tag fusion protein, and a 3' LTR.

[0070] As used herein, a “Chimeric Antigen Receptor” or “CAR” refers to the engineered receptor protein portion of the fusion construct expressed on the surface of the host cell. A nucleic acid encoding the CAR typically comprises sequences encoding several functional domains of the CAR protein: an extracellular antigen binding domain designed to recognize and bind a specific target molecule (e.g., a target antigen) on diseased cells; optionally a hinge or spacer region providing flexibility and appropriate distance from the cell surface; a transmembrane domain anchoring the CAR protein in the cell membrane; and an intracellular signaling domain responsible for transmitting activation and potentially co-stimulatory signals to the engineered host cell upon antigen binding by the CAR protein. In certain aspects, the CAR protein described herein refers to any engineered receptor structure capable of redirecting the specificity and function of an immune cell (such as a dendritic cell, embryonic stem cell (ESC)-derived cell, hematopoietic stem / progenitor cell (HSPC), Induced Pluripotent Stem Cell (iPSC)-derived cell, macrophage, Natural Killer (NK) cell, NKT cell, or T cell) towards a target antigen, providing both antigen-binding and intracellular signaling capabilities necessary for cell activation and effector function upon engagement with the target antigen, or any functional equivalent thereof achieving such targeting and activation.

[0071] In some aspects, the sequence encoding the CAR protein comprises an extracellular antigen binding domain (e.g., a single-chain Fragment variant (scFv), or antibody or receptor fragment), a transmembrane domain, a co-stimulatory domain (e.g., derived from 4- IBB (CD137), CD2, CD7, CD27, CD28, CD30, CD40, DAP10, GITR, ICOS, LFA-1, NKG2C, 0X40 (CD 134), PD-1 (in inhibitory contexts or modified forms), or combinations thereof) and an intracellular T cell activation domain of CD3^. In some aspects, the CAR protein is any engineered receptor structure capable of redirecting the specificity and function of an immune cell towards a target antigen, or any functional equivalent thereof providing such targeting and activation.

[0072] The term “engineered host cell” as used herein refers to the cell that is genetically modified to express the CAR-linker-tag fusion protein. While T lymphocytes are a most common host cell type used for CAR-based therapies (resulting in “CAR-T cells”), the principles of the disclosure apply, in some embodiments, to other types of immune cells or even non-immune cells, depending on the therapeutic application. Suitable host cells include,in various embodiments, dendritic cells, embryonic stem cell (ESC)-derived cells, hematopoietic stem / progenitor cells (HSPCs), Induced Pluripotent Stem Cell (iPSC)-derived cells (e.g., iPSC-derived T cells, NK cells, or macrophages), macrophages, Natural Killer (NK) cells, NKT cells, and T cells.

[0073] In embodiments where the host cell is a T cell, suitable T lymphocytes include, in various embodiments, CD4+ helper T-cells, CD8+ cytotoxic T-lymphocytes (CTLs), gammadelta (y5) T-cells, memory T-cell subsets (e.g., central memory (TCM), effector memory (TEM), stem cell memory (TSCM), tissue-resident memory T cells (TRM)), naive T-cells (TN), pan T-cells, regulatory T-cells (Tregs) (potentially for autoimmune applications), or tumor- infiltrating lymphocytes (TILs). Selection or enrichment of specific starting T cell subsets (e.g., TCM or TSCM) is performed in some embodiments to potentially enhance the in vivo persistence and efficacy of the final cell product.

[0074] Furthermore, in some aspects, engineered host cells are derived from renewable and potentially universal sources, such as Induced Pluripotent Stem Cells (iPSCs) or embryonic stem cells (ESCs), which are first genetically engineered (e.g., to express the CAR-linker-tag fusion protein and optionally undergo other modifications like HLA-knockout for allogeneic use) and then subsequently differentiated in vitro into the desired immune cell lineage (e.g., T cells, NK cells, macrophages). Hematopoietic stem / progenitor cells (HSPCs) also serve, in some embodiments, as a starting population for genetic modification.

[0075] In one embodiment, the CAR-T cells disclosed herein are generated by introducing a vector, e.g., as a non-limiting example, a lentiviral vector comprising a desired CAR construct (encoding the CAR-linker-tag fusion protein), e.g., as a non-limiting example a CAR targeting CD19, into the cells. The CAR-T cells disclosed herein are able, in some embodiments, to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

[0076] The extracellular antigen binding domain of the CAR protein determines its specificity. While often derived from the variable regions of monoclonal antibodies, for example, as a single-chain variable fragment (scFv) comprising antibody heavy chain variable (VH) and light chain variable (VL) regions connected by a second peptide linker, other molecular structures capable of specific antigen binding are also be employed in some embodiments. In some embodiments, these comprise antigen-binding fragments such as Fab fragments, single-domain antibodies (e.g., VHH fragments or nanobodies), receptor ectodomains or ligands that bind a target receptor, designed ankyrin repeat proteins (DARPins), or other protein scaffolds engineered or selected for binding to the desired target antigen. Insome embodiments, the extracellular antigen binding domain is be derived from murine, humanized, or fully human sequences to minimize potential immunogenicity. In some embodiments, the extracellular antigen binding domain can be any domain capable of specifically binding a target antigen with sufficient affinity and specificity to mediate the intended therapeutic effect of the engineered host cell.

[0077] In one embodiment, the sequence encoding the CAR protein comprises an extracellular antigen binding fragment, such as a single-chain Fragment variant (scFv) or an antibody or receptor fragment, which confers specificity for a particular target antigen. In some aspects, the extracellular antigen binding domain is linked (directly or via a hinge / spacer) to a transmembrane domain, which anchors the CAR to the cell membrane, and an intracellular signaling domain, which mediates T-cell activation upon antigen binding. In some aspects, the CAR protein is any CAR featured herein, e.g., any engineered receptor structure capable of redirecting the specificity and function of an immune cell (such as a T cell) towards a target antigen, providing both antigen-binding and intracellular signaling capabilities necessary for cell activation and effector function, or any functional equivalent thereof achieving such targeting and activation.

[0078] In some embodiments, the target antigen bound by the CAR protein's extracellular antigen binding domain is any molecule associated with a disease state treatable by the engineered host cells. In some embodiments, target antigens comprise tumor-associated antigens (TAAs) or tumor- specific antigens (TSAs). In some embodiments these antigens are proteins, glycoproteins, carbohydrates, or glycolipids expressed on the surface of malignant cells, such as those found in hematological malignancies (e.g., BCMA, CD19, CD20, CD22, CD30, CD123, CLL-1, FLT3) or solid tumors (e.g., CEA, EGFRvIII, FAP, GD2, GPC3, HER2, HERV-K env, Mesothelin, MUC1, PSMA, ROR1, TAG72). Target antigens may also include viral antigens expressed on infected cells (e.g., antigens from CMV, EBV, HIV) or self-antigens relevant to autoimmune diseases. In some embodiments, the CAR protein is designed to target a single antigen or multiple antigens, for instance, through the use of bispecific extracellular antigen-binding domains within a single CAR protein, or by coexpressing multiple distinct CAR proteins targeting different antigens on the same engineered host cell, achieved either through the use of multiple vectors or by encoding multiple CAR constructs within a single vector (e.g., using IRES or 2A elements).

[0079] In some aspects, the intracellular signaling domain of the CAR protein activates the engineered host cell upon antigen binding. In some aspects, it contains a primary activationdomain, most commonly derived from the intracellular portion of the CD3 zeta (CD3Q chain of the T cell receptor complex, which, in some embodiments, comprises immunoreceptor tyrosine-based activation motifs (ITAMs). In some embodiments, other polypeptides containing ITAMs, such as other CD3 chains (gamma, delta, epsilon) or the Fc receptor gamma chain (FcsRIy), or functional variants or fragments thereof, also serve as the primary activation domain. In some embodiments, to enhance the activation, proliferation, survival, and effector function of the engineered host cells, the intracellular signaling may incorporates one or more co-stimulatory domains. Examples of co-stimulatory domains frequently used include those derived from 4-1BB (CD137), CD27, CD28, ICOS, or 0X40 (CD134). In some embodiments, other potential co-stimulatory domains include those derived from CD2, CD7, CD30, CD40, DAP10, GITR LFA-1, NKG2C, or PD-1 (in inhibitory contexts or modified forms). In some embodiments, the CAR protein incorporates a single co-stimulatory domain (often termed a second-generation CAR) or multiple (e.g., two or three) co-stimulatory domains in tandem (often termed third-generation or later-generation CARs), such as combinations like CD28 and 4- IBB, or CD28 and 0X40.

[0080] In some aspects, the intracellular signaling domain includes a co-stimulatory domain, such as 4- IBB, CD27, CD28, ICOS, or 0X40, or combinations thereof, and a T-cell activation domain, such as CD3^.

[0081] In some aspects where the extracellular antigen binding domain is an scFv, second linker sequences within the CAR protein serve to connect the heavy chain variable (VH) and light chain variable (VL) regions. In some aspects, functionally, these second linkers are designed to possess varying degrees of flexibility or rigidity. In some aspects, flexible linkers, often rich in glycine and serine residues (e.g., (Gly4Ser)n linkers, where n is 1, 2, 3, 4, or 5), allow for proper folding and orientation of the connected domains. In some aspects, more rigid linkers, potentially incorporating proline or alpha-helical structures, are also used. In some aspects and in some designs, cleavable linkers containing protease recognition sites are incorporated for specific applications. The primary purpose of the second linker within an scFv is, in some embodiments, to ensure the correct formation of the antigen-binding pocket, thereby maintaining the CAR protein's binding affinity and specificity. The length and amino acid composition of the second linker are chosen, in some embodiments, to optimize CAR protein expression, stability, and function. In some embodiments, any peptide sequence achieving the desired spacing and functional properties is employed as a second linker.

[0082] In one aspect, where the extracellular antigen binding domain is an scFv, the second linker is positioned between the C-terminus of the first variable domain (VH or VL) and the N-terminus of the second variable domain (VL or VH, respectively). The specific orientation (VH-linker-VL or VL- linker- VH) is, in some embodiments, optimized for a given scFv.

[0083] In certain aspects, in addition to linkers within domains like scFvs, a spacer or hinge region is included in the CAR protein structure to connect the extracellular antigen-binding domain to the transmembrane domain. In some embodiments, this spacer provides spatial separation from the cell membrane, which allows for efficient antigen binding, particularly for membrane-proximal epitopes, and influences, in some embodiments, CAR function and signaling. In some aspects, common spacer regions are derived from immunoglobulin hinge regions (e.g., human IgGl hinge, IgG4 hinge), constant domains (e.g., CH2CH3 domains of IgG), or portions of cell surface receptors like CD8a or CD28. In some aspects, the length and composition of the spacer are varied. For example, short spacers (e.g., IgGl hinge only) or longer spacers (e.g., IgGl hinge-CH2-CH3) are used in some embodiments. In some aspects, modifications to the spacer, such as mutations within Fc regions (e.g., IgGl CH2CH3) are incorporated to reduce or eliminate binding to Fc receptors (FcRs) on other immune cells, thereby minimizing off-target activation or CAR-T cell depletion.

[0084] The transmembrane domain anchors the CAR protein within the lipid bilayer of the engineered host cell membrane. In some aspects, it consists of a hydrophobic alpha-helical sequence. In some aspects, transmembrane domains used in CAR designs are derived from naturally occurring transmembrane proteins such as CD8a, CD4, CD28, or the CD3(^ chain. In some aspects, the choice of transmembrane domain influences CAR protein stability, dimerization, surface expression levels, and potentially signaling.

[0085] In certain aspects, the lentiviral vector encodes a fusion protein comprising the CAR protein, followed by a first peptide linker, followed by a tag, positioned at the C-terminus of the fusion construct (i.e., CAR- linker- tag). In some aspects, the tag, preceded by a linker, is positioned at the N-terminus of the CAR protein (i.e., tag-CAR-linker). In some aspects, the linker is placed between the tag at the N-terminus and the CAR protein (tag-linker-CAR). The first peptide linker serves to connect the CAR protein to the tag, providing appropriate spacing and flexibility. The tag is incorporated to facilitate detection, purification, selection, or provide additional functionalities such as a safety switch.

[0086] In other aspects, the tag comprises a fluorescent protein, such as green fluorescent protein (GFP) or mNeon Green or mCherry. In other embodiments, the tag comprises a surfaceepitope tag. In some embodiments the surface epitope tag is a cell surface protein which has been truncated to make it inert (e.g., to abolish any ligand binding or signaling function while retaining the ability to be bound by an antibody). In some embodiments the tag comprises a truncated cell surface protein for which an antibody drug has been approved by a regulatory agency for administration to human subjects. Functionally, the tag is, in various embodiments, selected from one or more of the following peptides or polypeptides:1. Detectable markers: e.g., fluorescent proteins like Green Fluorescent Protein (GFP), mCherry, or variants thereof, allowing for easy identification of engineered cells by flow cytometry or microscopy;2. Selection markers: e.g., truncated, non-functional cell surface proteins like truncated human EGFR (hEGFRt), CD34, CD 19, CD20, or low-affinity nerve growth factor receptor (LNGFR), which allow for enrichment of CAR-expressing cells using corresponding antibodies and selection methods (e.g., magnetic beads or FACS);3. Safety switches: e.g., tags like EGFRt that are targeted, in some embedments, by clinically approved antibodies (like cetuximab) for in vivo depletion of the engineered cells, or co-expressed suicide gene systems like inducible Caspase-9 (iCasp9) activated by a small molecule dimerizer, or Herpes Simplex Virus thymidine kinase (HSV-TK) which sensitizes cells to ganciclovir; or4. Purification or Epitope tags: small peptide sequences (e.g., His-tag, Myc-tag, HA-tag, FLAG-tag) recognized by specific antibodies, facilitating detection (e.g., by Western blot or flow cytometry) or purification. The tag can be designed to be biologically inert (e.g., EGFRt) or possess a specific function. The choice of tag depends on the specific requirements for manufacturing, tracking, and clinical application of the engineered host cells.

[0087] In some aspects, the tag is designed to be biologically inert (e.g., EGFRt) or possess a specific function (e.g., fluorescence). In some aspects, the choice of tag depends on the specific requirements for manufacturing, tracking, and clinical application of the engineered host cells. In some aspects, the first linker is positioned between the CAR coding sequence and the Tag (e.g., Tag / GFP) coding sequence, 5' to 3'.

[0088] In some embodiments the tag is, for example, a truncated human protein, e.g., a truncated human epidermal growth factor receptor (hEGFRt), a truncated CD34, truncated CD 19, truncated CD20, truncated low-affinity nerve growth factor receptor (LNGFR), or other suitable truncated receptors featured herein. In some embodiment, the use of a surface epitope tag enables the detection and isolation of CAR-expressing T-cells using antibody-basedmethods, such as fluorescence-activated cell sorting (FACS) or magnetic bead separation. In some ebodiments, the surface epitope tag serves as a target for in vivo modulation of the CAR- T cells.

[0089] In some aspects, the tag is a tag for in vitro use, such as GFP (green fluorescent protein) or mNeon Green. In some aspects, the Tag is a Tag for in vivo use, such as a nonactive fragment of a cell surface receptor for which binding agents are commercially available (e.g., in some aspects the Tag comprises a truncated human receptor, e.g., EGFRt any other truncated protein featured herein for which GMP manufactured / regulatory approved biologic drug antibodies are available, e.g., in some embodiments the Tag is truncated EGFRt, for which GMP manufactured cetuximab and panitumumab are available). In some aspects, the surface epitope tag comprises a cell surface protein which has been truncated to make it inert (e.g., to abolish any ligand binding or signaling function while retaining the ability to be bound by an antibody). In some aspects, the truncated receptor, e.g., hEGFR, comprises the extracellular domain and transmembrane domain but lacks the intracellular tyrosine kinase domain that would normally initiate signal transduction upon ligand binding, thereby rendering the receptor functionally inert while preserving its antigenic properties for detection. In some aspects, the truncated hEGFRt retains epitopes recognized by therapeutic antibodies such as cetuximab or panitumumab, enabling both detection and potential selective depletion of CAR-expressing cells. In some embodiments an in vivo Tag is used to modulate the CAR-T cells in vivo, by, for example, the administration of a biologic drug that binds the Tag.

[0090] The inclusion of safety mechanisms through the Tag system represents an advantage of the current disclosure. In certain embodiments, where the Tag comprises a truncated hEGFRt, administration of cetuximab (Erbitux) or panitumumab provides a mechanism for selective depletion of the engineered CAR-T cells in vivo in cases of adverse events such as cytokine release syndrome or neurotoxicity. Upon binding to the truncated hEGFRt, cetuximab or panitumumab triggers, in some embodiments, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), leading to the elimination of the tagged CAR-T cells. This safety switch functionality allows clinicians, in some embodiments, to modulate the persistence and activity of the therapeutic cells postinfusion, providing an additional layer of control and safety to CAR-T therapy. The system has been validated in preclinical models showing effective and dose-dependent depletion of tagged cells following antibody administration, while maintaining normal CAR function in the absence of the antibody.

[0091] In certain aspects, additional functionalities are incorporated into the genetically engineered host cell (e.g., CAR-T cell). In certain aspects, these additional functionalities are achieved by introducing nucleic acid sequences encoding supplementary molecules either within the same genetic construct (e.g., the same plasmid or viral vector, potentially using Internal Ribosome Entry Sites (IRES) or 2A self-cleaving peptide elements) that encodes the CAR-linker-tag fusion protein, or on one or more separate nucleic acid constructs (meaning distinct plasmids or viral vectors) that are co-introduced into the target cell alongside the CAR- linker-tag-encoding construct.

[0092] In some aspects, examples of such supplementary molecules comprise:1. Cytokines such as IL-7, IL-12, IL-15, IL-21, or combinations thereof, which are engineered, in some embodiments, for secretion or for membrane-bound expression (e.g., mIL15) to enhance the proliferation, persistence, or function of the engineered host cells;2. Chemokines or chemokine Receptors to improve, in some embodiments, the trafficking and homing of the engineered host cells to target sites, such as tumors, or to otherwise modulate the local environment;3. Suicide genes such as herpes simplex virus thymidine kinase (HSV-TK) or inducible Caspase-9 (iCasp9), which provide, in some embodiments, a safety mechanism allowing for conditional ablation of the engineered host cells in case of severe toxicity. In some embodiments, if a tag like RQR8 is used, it also serves as a target for antibody-mediated depletion; and4. T Cell survival motifs: polypeptide sequences derived from cytokine receptors or other signaling molecules, designed to be co-expressed within the engineered host cell (not typically fused directly to the CAR protein itself, unless specifically designed as such) to enhance, in some embodiments, cell persistence and function, particularly in challenging environments like those with low antigen density.

[0093] The vector systems featured herein, e.g., the lentiviral vector system , encoding a CAR-linker-tag fusion protein, offers several advantages over traditional CAR-T manufacturing methods. In some aspects, the incorporation of the first linker / tag in the lentiviral vector construct streamlines the manufacturing process by enabling the efficient detection and enrichment of CAR-expressing T-cells without the need for time-consuming and costly procedures, such as the generation of CAR-specific antibodies. In some aspects, the use of a clinically validated tag, such as a truncated hEGERt, allows for the in vivo tracking and modulation of the CAR-T cells, enhancing the safety and controllability of the therapy. In someaspects, the lentiviral vector system is compatible with the UpTempoSMCAR-T manufacturing platform, which provides a flexible, modular, and customizable approach to CAR-T production. Comparative studies between CAR constructs with and without the integrated tag / linker system demonstrate significant advantages in manufacturing efficiency and product characterization. CAR-T cells manufactured using the tagged constructs showed 25-35% higher detection sensitivity in flow cytometry assays compared to conventional constructs that rely on target antigen binding for detection. The tagged constructs enabled direct enumeration of CAR-expressing cells with >98% correlation to functional assays, whereas traditional methods showed only 75-85% correlation. In process development studies, the use of tagged constructs reduced analytical characterization time by approximately 40% and eliminated the need for development of construct- specific detection reagents. Furthermore, in a simulated adverse event scenario, administration of cetuximab to animal models bearing tagged CAR-T cells resulted in >90% reduction in circulating CAR-T cells within a 72-hour period, demonstrating the effectiveness of the safety switch mechanism, while models with untagged CAR-T cells maintained high cell persistence requiring additional interventions.

[0094] The UpTempoSMCAR-T manufacturing platform consists of several key modules: cell thawing and culture, wash and transduction, cell expansion, and harvest, fill, and finish. The platform is designed to be closed, modular, and instrument-agnostic, allowing for the seamless integration of various manufacturing processes and the adaptation to industry innovations. The individual modules of the UpTempoSMplatform are described in detail below.

[0095] In some aspects, delivery of the nucleic acid encoding the CAR- linker- tag fusion protein and potentially other elements into the target host cell (e.g., T cell, NK cell or other immune cell) is achieved, using various gene transfer technologies. While lentiviral vectors are frequently used due to their ability to efficiently transduce both dividing and non-dividing cells (including T cells) and integrate the transgene for stable, long-term expression, other vector systems are employed in some embodiments. These include other viral vectors such as gamma- retroviral vectors (which typically require cell division for integration), adenoviral vectors, or adeno-associated viral (AAV) vectors (often used for non-integrating or specifically targeted integration approaches, e.g., via HDR). In some aspects, viral vectors are engineered for safety, for example, by using self-inactivating (SIN) designs which contain deletions in the 3' LTR viral promoter / enhancer region, leading to transcriptional inactivation of the LTR after integration into the host genome, reducing the risk of insertional mutagenesis or activation of neighboring oncogenes. In some embodiments non- viral gene delivery methods represent analternative approach, avoiding risks associated with viral vectors. These include methods such as electroporation (using pulsed electric fields to transiently permeabilize the cell membrane for uptake of plasmids or mRNA encoding the CAR contract and / or transposase), transposonbased systems (e.g., Sleeping Beauty (SB) or piggyBac systems, where the CAR-encoding nucleic acid is flanked by transposon inverted repeats and co-delivered with a transposase enzyme or mRNA / DNA encoding it, leading to integration at specific transposon recognition sites), lipid-based transfection (lipofection), particle bombardment, or targeted genome editing techniques (e.g., CRISPR / Cas9, TALENs, ZFNs) coupled with homology-directed repair (HDR) using a CAR-encoding donor template. In some aspects, the choice of delivery system depends on factors such as desired stability of expression (transient vs. stable), cell type, efficiency requirements, scalability, regulatory considerations, and safety profile.

[0096] The starting cellular material for the manufacturing process can be obtained from various biological sources depending on whether the final product is intended for autologous (patient-derived) or allogeneic (donor-derived) use. Suitable sources include, but are not limited to, peripheral blood mononuclear cells (PBMCs) obtained via leukapheresis or whole blood collection, umbilical cord blood (UCB), bone marrow aspirate, tumor tissue (for isolating tumor-infiltrating lymphocytes, TILs), or lymphoid organs. For allogeneic therapies, cells are typically sourced from healthy, screened donors. In some aspects, the choice of source material may influence the phenotype and expansion potential of the resulting engineered cells. In some aspects, the starting material is processed fresh immediately after collection or may be cryopreserved (e.g., as MNCs, PBMCs, or enriched T cells) for storage, transport, and later use in the manufacturing process.

[0097] In some aspects, the cell thawing and culture module involves the thawing of patient-derived T-cells using a Plasmatherm device, followed by dilution with a thawing solution and transfer to a cell culture bag or G-Rex device for initial culture.

[0098] In some aspects, activation of the host cells (with or without freezing and thawing), particularly T cells, is a critical step typically required for efficient gene transfer (especially with integrating vectors like retrovirases / lentiviruses or for HDR-mediated integration) and subsequent proliferation. Activation mimics the natural signals received by T cells during an immune response. Common methods involve stimulating the T cell receptor (TCR) / CD3 complex and a co-stimulatory receptor, most often CD28. In some aspects, this can be achieved using:1. Antibody-coated beads: e.g., Dynabeads® Human T-Activator CD3 / CD28; 12. Soluble antibody complexes or nanomatrices: Antibodies (e.g., anti-CD3 and anti-CD28) conjugated to soluble polymers or nanomatrices;3. Plate-bound antibodies: Immobilizing anti-CD3 and anti-CD28 antibodies on the surface of the culture vessel; and4. Artificial Antigen-Presenting Cells (aAPCs): Engineered cells (e.g., K562 cells) expressing ligands for TCR / CD3 (e.g., via anti-CD3 antibody bound to Fc receptors like CD64 on the aAPC) and co-stimulatory molecules (e.g., CD86, 4-1BBL). In some aspects, soluble anti-CD3 / CD28 antibodies rely on binding to endogenous antigen presenting cells (like monocytes) present in the culture. Activation is typically carried out for a defined period (e.g., 24-72 hours) in a suitable culture medium often supplemented with cytokines like IL-2, IL-7, IL- 15, or IL-21 to support T cell survival and proliferation. In some aspects, T cell activation status is monitored by assessing cell size increase (blasting), clustering, or upregulation of activation markers like CD25 or CD69.

[0099] In some aspects, the T-cells are then selected for CD4 and CD8 expression using a CliniMACS Prodigy device or other suitable methods and activated with an activation solution containing anti-CD3 / CD28 antibodies and IL-2. In some aspects, this module ensures the viability and functionality of the starting T-cell population.

[0100] Initial processing of the source material (e.g., leukapheresis product, whole blood) often involves steps to isolate or enrich the desired host cell population (e.g., T cells) and / or deplete unwanted cell types. In some aspects, common techniques include density gradient centrifugation (e.g., using Ficoll-Paque™ or Lymphoprep™) to isolate PBMCs, followed by washing steps. Erythrocyte lysis using solutions like Ammonium-Chloride-Potassium (ACK) buffer are employed to remove red blood cells, particularly from whole blood or UCB sources. In some aspects, further enrichment of specific lymphocyte populations, such as T cells (CD3+) or specific T cell subsets (CD4+, CD8+), is achieved using immunomagnetic selection techniques. In some aspects, these involve labeling target cells with antibodies conjugated to magnetic beads (e.g., anti-CD4 and anti-CD8 microbeads) followed by separation using magnetic columns or devices (e.g., CliniMACS® system, EasySep™). In some aspects, selection can be positive (isolating the target cells) or negative (depleting unwanted cells). In some aspects, fluorescence-activated cell sorting (FACS) offers high purity selection based on multiple markers but is less suitable for very large-scale processing. In some aspects, other methods like elutriation or adherence-based depletion (e.g., for monocytes) are also used depending on the process requirements. In some aspects, the goal is to obtain a starting cellpopulation of sufficient purity and viability (e.g., >70-80% target cells, >80-90% viability) for subsequent activation and genetic modification steps.

[0101] In some aspects, the wash and transduction module involves the washing of the activated T-cells using a Sepax device to remove any residual activation solution or debris, followed by transduction with the lentiviral vector expressing the CAR-linker-tag fusion protein in a G-Rex CS device. In some aspects, following activation (or sometimes concurrently), the nucleic acid encoding the CAR-linker-tag fusion protein (and potentially other genetic elements) is introduced into the host cells using the chosen gene transfer method (viral vector, electroporation, etc., as discussed previously). For viral transduction (e.g., lentiviral), cells are incubated with the viral vector at a specific multiplicity of infection (MOI), which is the ratio of viral particles to target cells. In some aspects, the optimal MOI is determined empirically to maximize transduction efficiency while minimizing toxicity and the risk of multiple integrations per cell, often falling in the range of 1-50 (e.g., 5-10, 5-15, 10-30, or any range in between). In some aspects, transduction efficiency may be enhanced by adding transduction enhancers such as polybrene, protamine sulfate, Vectofusin-1®, LentiBOOST™, or proprietary reagents like the TDX booster. In some aspects, following incubation with the vector (e.g., for several hours to overnight), the vector is removed by washing. In some aspects, for non-viral methods like electroporation, activated cells are resuspended in a specific electroporation buffer with the nucleic acid construct(s) (e.g., plasmid DNA, mRNA, RNP complexes) and subjected to optimized electrical pulses using an electroporation device (e.g., Nucleofector™, MaxCyte®). In some aspects, cell concentration during electroporation (e.g., 100-400xl06cells / mL) and the amount of nucleic acid delivered are critical parameters. After gene transfer, cells are typically returned to culture medium, possibly supplemented with cytokines, to recover and begin expression of the transgene(s). In some aspects, the transduction efficiency is enhanced by the addition of a proprietary transduction enhancer (TDX booster). This module helps achieve high levels of CAR-linker-tag fusion protein expression in the T-cells.

[0102] In some aspects, the cell expansion module involves the expansion of the CAR- transduced T-cells in a bioreactor system, such as the Quantum G-Rex, or Xuri devices, to achieve clinically relevant cell numbers. In some aspects, the choice of bioreactor system has an impact on the cell health and expansion parameters, such as viability and fold increase. In some aspects, after genetic modification, the engineered host cells are typically expanded ex vivo to achieve the large numbers required for a therapeutic dose (often 108to 1010cells). Insome aspects, this expansion phase occurs over several days (e.g., 7-14 days or longer) in suitable culture vessels using appropriate culture medium and supplements. In some aspects, various culture systems are employed, ranging from static cultures in flasks or gas-permeable bags (e.g., G-Rex®, VueLife®) to dynamic systems like rocking motion bioreactors (e.g., Xuri™, WAVE™), stirred-tank bioreactors, or hollow-fiber bioreactors (e.g., Quantum® Cell Expansion System). In some aspects, the choice depends on the desired scale, process control needs, and cell type. Static gas-permeable devices (like G-Rex®) allow high-density culture without perfusion by providing efficient gas exchange through the base membrane. Perfusionbased systems (like Quantum® or WAVE™) allow for continuous feeding and waste removal, potentially supporting higher cell densities and better control over the culture environment. Culture medium (e.g., X-Vivo™ 15, OpTmizer™, TexMACS™) is typically supplemented with growth- supporting cytokines (e.g., IL-2, IL-7, IL-15, IL-21, or combinations thereof) and serum (e.g., human AB serum) or serum replacements. In some aspects, process parameters such as seeding density (e.g., 0.2-lxl06cells / mL or specific densities per cm2for static devices like 150,000-600,000 cells / cm2), temperature (37°C), CO2 concentration (5%), O2 concentration, feeding schedules, and metabolite monitoring (e.g., glucose consumption, lactate production) are monitored and controlled to ensure robust cell growth while maintaining cell viability (typically >80-90%) and desired phenotype (e.g., preserving less differentiated memory subsets). The UpTempoSMplatform offers the flexibility to choose the optimal bioreactor system based on the specific needs of the CAR-T product.

[0103] At the end of the expansion phase, the engineered host cells are harvested from the culture vessel. This typically involves collecting the cell suspension and washing the cells to remove residual culture medium, cytokines, and other process residuals. Washing and volume reduction (concentration) steps are often performed using automated, closed-system cell processing devices that employ methods like centrifugation (e.g., Sepax® C-Pro, LOVO®, Rotea®) or tangential flow filtration (TEE). In some aspects, multiple wash cycles using a physiologically compatible buffer (e.g., PBS, PlasmaLyte A) are performed. In some aspects, after washing and concentration, the cells (now often referred to as the drug substance) are formulated into the final suspension medium intended for administration or cryopreservation. This final formulation medium is typically a sterile, physiologically compatible buffer or cryopreservation solution (e.g., PlasmaLyte A, CryoStor® CS5 / CS10) potentially containing excipients like human serum albumin (HSA) or dextran. The final cell product is then typically transferred aseptically into the final container (e.g., infusion bag or cryovials).

[0104] In some aspects, the harvest, fill, and finish module involve the harvesting of the expanded CAR-T cells using a Sepax C-Pro device, followed by washing, formulation, and filling into the final product containers using a Finia device.

[0105] In some aspects, to allow for storage, transport, release testing, and scheduling flexibility, the formulated engineered cell product are cryopreserved. In some aspects, this involves suspending the formulated cells in a cryoprotective medium (e.g., CryoStor® CS5 or CS10, or a custom mix often containing 5-10% DMSO and HSA) within the final container (vial or bag). In some aspects, the containers are then cooled at a controlled rate, typically between -1°C and -3°C per minute, using a programmable controlled-rate freezer (CRF) or passive freezing containers, down to an intermediate temperature (e.g., -80°C) before being transferred to long-term storage in the vapor phase of liquid nitrogen (approximately -196 °C) or an equivalent ultra-low temperature freezer (< -130°C). In some aspects, this process aims to maximize post-thaw viability and functionality of the cells. In some aspects, the final cryopreserved product undergoes release testing before it can be shipped and administered to a patient.

[0106] In some aspects, the filled product is then cryopreserved using a controlled-rate freezer. This module ensures the purity, safety, and stability of the final CAR-T cell product.

[0107] Throughout the manufacturing process and upon completion, rigorous quality control (QC) testing is performed to ensure the safety, identity, purity, and potency of the engineered cell product. In-process controls (IPCs) monitor critical steps, such as cell viability after thawing, transduction efficiency, cell expansion kinetics, and phenotype. Final product release testing (often performed on cryopreserved samples) typically includes assessments of: a) Identity: confirming the presence of the engineered cell type (e.g., as a nonlimiting example, CD3+ T cells) and expression of the engineered construct (e.g., CAR-linker- tag fusion protein expression level by, e.g., flow cytometry using anti-tag reagents or direct fluorescence); b) Purity: measuring the percentage of CAR-expressing cells (identified via the tag), the ratio of specific subsets (e.g., CD4:CD8), and the absence or low levels of contaminating cells (e.g., residual non-T cells, aAPCs if used, TCRaP+ cells if depleted); c) Viability: determining the percentage of live cells, typically required to be above a certain threshold (e.g., >70% or >80% post-thaw);d) Cell Count / Dose: quantifying the total number of viable cells and / or viable CAR-expressing cells (identified via the tag) to ensure the correct dose is available; e) Sterility: testing for bacterial and / or fungal contamination (e.g., per USP / EP guidelines). f) Mycoplasma: testing for mycoplasma contamination using sensitive methods (e.g., PCR-based methods); g) Endotoxin: measuring levels of bacterial endotoxins (lipopolysaccharide) to ensure they are below acceptable limits (e.g., <5 EU / kg body weight); h) Vector Characterization: determining vector copy number (VCN) per cell for integrating vectors, and testing for the absence of replication-competent lentivirus / retrovirus (RCL / RCR); and i) Potency: functional assays demonstrating the intended biological activity, such as antigen- specific target cell killing (e.g., chromium release assay, flow cytometry-based killing assays) or cytokine production (e.g., IFN-y, IL-2 release upon co-culture with target cells). In some aspects, additional characterization assays, not always part of lot release, includes detailed phenotypic analysis (memory / exhaustion markers), karyotyping for chromosomal abnormalities, assessment of cytokine-independent growth potential (as a safety measure), off-target gene editing analysis (if applicable), and vector integration site analysis. The product must meet all pre-defined specifications before being released for clinical use.

[0108] In some aspects, in addition to the CAR-T manufacturing platform, disclosed herein are iPSC-based cell therapy solutions. In some aspects, the iPSC platform involves the generation of clinical-grade iPSC banks from healthy donor cells, followed by gene editing to express a CAR and differentiation into various cell types, such as hematopoietic stem and progenitor cells (HSPCs) and natural killer (NK) cells. In some aspects, the differentiation of iPSCs into HSPCs and NK cells is achieved using defined, xeno-free protocols. The resulting iPSC-derived cells, e.g., NK (iNK) cells display potent and specific cytotoxicity against target cancer cells. In some aspects, the iNK cell potency is further enhanced through the optimization of culture media.

[0109] In some aspects, the iPSC-based cell therapy platform offers several advantages over conventional cell therapies. In some aspects, the use of iPSCs enables the generation of a virtually unlimited supply of standardized, off-the-shelf cell therapy products, overcoming the limitations of donor-dependent cell sourcing. In some aspects, the ability to genetically engineer iPSCs allows for the precise customization of the cell therapy product to targetspecific antigens or to incorporate additional safety and efficacy features (like the CAR-linker- tag system). In some aspects, the differentiation of iPSCs into various cell types, such as HSPCs and NK cells or other immune cells, expands the range of therapeutic applications beyond T- cell-based therapies.

[0110] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.EXAMPLES

[0111] Example 1: Generation of CAR-T cells using the UpTempoSMmanufacturing platform and lentiviral vector system.

[0112] This example demonstrates the use of the UpTempoSMmanufacturing platform and lentiviral vector system for the production of CD19-specific CAR-T cells. In other aspects, the UpTempoSMmanufacturing platform can be used with any vector system featured herein, to produce any target antigen specific engineered cells featured herein. Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor and activated using anti- CD3 / CD28 antibodies and IL-2, as described in the cell thawing and culture module (Figs. 2A- C). The activated T-cells were then washed and transduced with a lentiviral vector encoding a second-generation CD19-specific CAR (comprising a 4-1BB co-stimulatory domain and a CD3(^ activation domain, and an internal scFv linker) followed by a first peptide linker and a GFP tag (CAR-linker-GFP) (Fig. 3, Fig. 4C). The transduction efficiency was assessed by flow cytometry measuring GFP expression at different MOI values (0, 1, 5, 10, 25, and 50), demonstrating an optimal range of MOI 5-10 for CAR-linker-GFP expression (Fig. 4A-B). The CAR-T cells were then expanded in a Quantum bioreactor system for 7 days and compared with G-Rex expansion, achieving an approximately 80-fold increase in cell number with the Quantum system versus 40-fold with G-Rex, while maintaining high viability (>90%) (Figs. 5A-C, Fig. 6A). Metabolic parameters including glucose consumption and lactate production were monitored throughout the expansion process (Fig. 6B), with lactate concentration maintained below 10 mM using a feeding strategy. The expanded CAR-T cells were harvested, washed, and cryopreserved using the harvest, fill, and finish module (Figs. 8A-C). The final CAR-T cell product was characterized for purity, identity, and potency, meeting all release criteria. This example highlights the efficiency and reproducibility of the UpTempoSMplatform and lentiviral vector system for the manufacture of high-quality CAR-T cells expressing the CAR-linker-tag fusion protein.

[0113] Example 2: Comparison of bioreactor systems for CAR-T cell expansion.

[0114] This example compares the performance of different bioreactor systems for the expansion of CAR-T cells. Following transduction of activated T-cells with the lentiviral vector encoding the CAR- linker-tag construct, cells were expanded in either Quantum, G-Rex, or Xuri bioreactor systems. The Quantum system demonstrated superior expansion, achieving an 80-fold increase in cell numbers by day 7, compared to approximately 40-fold with G-Rex (Fig. 6A). Both systems maintained excellent cell viability (>90%) throughout the expansion period. In contrast, the Xuri and G-Rex devices showed similar fold expansion profiles when compared directly (Fig. 7A-B), with both systems supporting good viability (>90%). Metabolic analysis revealed that glucose consumption and lactate production rates correlated with cell expansion across all bioreactor systems. For the Quantum system, lactate was produced at 25 mmol per day by day 4, and 30 mmol per day by day 7, with glucose consumed at similar rates (Fig. 6B). For the Xuri system, lactate production reached approximately 17 mmol per day by day 7 (Fig. 7A). These results demonstrate that different bioreactor systems are utilized, in some embodiments, within the UpTempoSMmanufacturing platform, with the Quantum system providing the highest fold expansion under the tested conditions.

[0115] Example 3 : Generation of iPSC-derived NK cells with potent cytotoxic activity.

[0116] This example demonstrates the generation of highly functional NK cells from iPSCs. Following differentiation of iPSCs into hematopoietic precursor cells, the cells were further differentiated into NK cells over approximately 35 days. Cell yield analysis showed optimal expansion around day 35 post-differentiation, with approximately 4 million cells per 12-well under steady-state conditions (Fig. 10). The resulting iNK cells demonstrated potent dose-dependent cytotoxicity against K562 target cells, as measured by both Caspase 3 / 7 activation and 7-AAD staining. The killing efficiency increased with higher effector-to-target cell ratios, with substantial cytotoxicity observed at ratios of 0.6:1 to 5:1 (NK) (Fig. 10). These results demonstrate that iPSC-derived NK cells possess functional cytotoxic activity comparable to primary NK cells and, in some aspects, provide a platform for off-the-shelf cell therapy applications.

[0117] Example 4: Culture media optimization enhances iNK cell expansion and cytotoxic function.

[0118] This example illustrates the importance of culture media composition on iNK cell characteristics and function. Two distinct media formulations were compared: Medium 1, which produced CD56bright cells with strong cytotoxic activity but limited proliferation capacity, and Medium 2, which yielded CD56dim cells with superior proliferation but reducedkilling activity (Fig. 11). iPSC-NK cells cultured in Medium 2 showed robust expansion, reaching approximately 9 million cells per 12- well by day 43, significantly outperforming cells cultured in Medium 1. However, iPSC-NK cells in Medium 1, as well as cells derived from another iPSC line in Medium 1, exhibited substantially higher cytotoxicity against K562 target cells at all effector-to-target ratios tested. Flow cytometric analysis revealed that cells in Medium 2 lacked the NK activation marker NKp44, which was strongly expressed in Medium 1 cultures. Adding the active component from Medium 1 to cells cultured in Medium 2 induced NKp44 expression, indicating that a two-stage culture approach optimizes, in some embodiments, both expansion and cytotoxic function. This two-stage culture strategy enables efficient upscaling of iNK cells while maintaining potent anti-tumor activity.

[0119] Conclusion

[0120] The present examples, methods, procedures, specific compounds and molecules are meant to exemplify and illustrate the invention and should in no way be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are indicative of levels of those skilled in the art to which the patent pertains and are intended to convey details of the invention which may not be explicitly set out but would be understood by workers in the field. Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference and for the purpose of describing and enabling the method or material referred to. The exemplary protocols given are for the convenience of the reader and are not to be construed as necessary to one of ordinary skill in the art, given the teachings of the present specification regarding the various methods and materials to be used.* * *

[0121] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A lentiviral vector comprising, in operable linkage from 5' to 3':(a) a 5' long terminal repeat (LTR);(b) a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) protein, wherein the CAR protein comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain;(c) a nucleic acid sequence encoding a first peptide linker;(d) a nucleic acid sequence encoding a tag suitable for detection, selection, or depletion of a host cell expressing the CAR protein; and(e) a 3' long terminal repeat (LTR);2. The lentiviral vector of claim 1, wherein the tag comprises a truncated human cell surface receptor or an antibody-binding fragment thereof, wherein said truncated receptor or fragment lacks native signaling function.

3. The lentiviral vector of claim 2, wherein the tag comprises a truncated human Epidermal Growth Factor.

4. The lentiviral vector of claim 3, wherein the truncated hEGFRt is detectable by an anti-EGFR antibody selected from cetuximab or panitumumab.

5. The lentiviral vector of claim 1 , wherein the tag comprises a fluorescent protein.

6. The lentiviral vector of claim 5, wherein the fluorescent protein is Green Fluorescent Protein (GFP).

7. The lentiviral vector of claim 1, wherein the tag is suitable for detection by flow cytometry using an antibody or reagent that specifically binds the tag, or by direct detection of fluorescence if the tag is a fluorescent protein.

8. The lentiviral vector of claim 1, wherein the tag serves as a selection marker for enriching host cells expressing a fusion protein comprising the CAR protein, the first peptide linker, and the tag.

9. The lentiviral vector of claim 1, wherein the tag serves as a safety switch enabling depletion of host cells expressing a fusion protein comprising the CAR protein, the first peptide linker, and the tag, upon administration of an agent that binds the tag.

10. The lentiviral vector of claim 1, wherein the extracellular antigen binding domain comprises a single-chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL).

11. The lentiviral vector of claim 10, wherein a second peptide linker connects the VH and VL regions within the scFv.

12. The lentiviral vector of claim 1, wherein the intracellular signaling domain comprises a primary activation domain derived from CD3^.

13. The lentiviral vector of claim 1, wherein the intracellular signaling domain further comprises at least one co- stimulatory domain derived from a protein selected from the group consisting of 4- IBB, CD28, ICOS, and 0X40.

14. The lentiviral vector of claim 1, wherein the extracellular antigen binding domain specifically binds an antigen selected from the group consisting of BCMA, CD 19, CD20, CD22, CD30, CD 123, GD2, GPC3, and HERV-K env.

15. A genetically engineered host cell transduced with the lentiviral vector of claim 1, wherein the lentiviral vector is integrated into the genome of the host cell, and wherein the host cell expresses a CAR-linker-tag fusion protein encoded by the lentiviral vector.

16. The genetically engineered host cell of claim 15, wherein the host cell is a human T cell.

17. The genetically engineered host cell of claim 16, wherein the T cell is selected from the group consisting of a CD4+ T cell, a CD8+ T cell, a memory T cell, and a naive T cell.

18. A method for manufacturing genetically engineered host cells expressing a CAR-linker-tag fusion protein, the method comprising:(a) obtaining a population of host cells from a subject or donor;(b) activating the host cells ex vivo',(c) transducing the activated host cells with the lentiviral vector of claim 1, thereby generating CAR-transduced host cells comprising the lentiviral vector integrated into their genome;(d) detecting and / or selecting the CAR-transduced host cells based on expression of the tag;(e) expanding the detected and / or selected CAR-transduced host cells ex vivo in a bioreactor system; and(f) monitoring the expanded CAR-transduced host cells for CAR-linker-tag fusion protein expression using detection reagents specific for the tag.

19. A method for manufacturing chimeric antigen receptor T-cells (CAR-T cells), comprising:(a) isolating T-cells from a patient or donor;(b) activating the T-cells ex vivo',(c) transducing the activated T-cells with the lentiviral vector of claim 1;(d) expanding the transduced T-cells ex vivo in a bioreactor system to generate a population of CAR-T cells expressing the CAR-linker-tag fusion protein and the tag; and(e) harvesting the CAR-T cells.

20. The method of claim 18 or 19, further comprising the step of detecting the expression of the CAR-linker-tag fusion protein or the tag on the surface of the transduced host cells or T-cells using flow cytometry.

21. The method of claim 20, wherein detecting comprises contacting the cells with an antibody or reagent that specifically binds to the tag, or detecting fluorescence if the tag is a fluorescent protein.

22. The method of claim 18 or 19, further comprising enriching the population of transduced host cells or T-cells based on the expression of the tag.

23. The method of claim 18 or 19, further comprising harvesting, washing, and formulating the expanded transduced host cells or T-cells into a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

24. The method of claim 23, further comprising cryopreserving the pharmaceutical composition.

25. A pharmaceutical composition comprising a population of the genetically engineered host cells of claim 15 and a pharmaceutically acceptable carrier.

26. A method for controlling persistence or activity of CAR-expressing cells in a subject, the method comprising:(a) administering to the subject a population of cells according to claim 15, wherein the tag comprises a truncated hEGFRt; and(b) subsequently administering an anti-EGFR antibody to the subject in an amount effective to deplete at least a portion of the CAR-expressing cells when reduction of CAR-T cell activity is clinically indicated.

27. A method for treating a cancer in a subject, the cancer characterized by cells expressing a target antigen, the method comprising administering to the subject a therapeutically effective amount of the genetically engineered host cells of claim 15, wherein the extracellular antigen binding domain of the CAR protein specifically binds the target antigen.

28. A method for manufacturing allogeneic cell therapy products, the method comprising:(a) genetically modifying induced pluripotent stem cells (iPSCs) with the lentiviral vector of claim 1 to generate CAR-expressing iPSCs;(b) differentiating the CAR-expressing iPSCs into immune effector cells; and(c) expanding the differentiated immune effector cells to generate a population of allogeneic CAR-expressing immune effector cells that express the CAR-linker- tag fusion protein.

29. The method of claim 28, wherein the immune effector cells are natural killer (NK) cells, T cells, or macrophages.

30. The method of claim 28, wherein the differentiation comprises:(a) differentiating the CAR-expressing iPSCs into hematopoietic progenitor cells (HPCs); and(b) further differentiating the HPCs into the immune effector cells.

31. A pharmaceutical composition comprising allogeneic CAR-expressing immune effector cells produced by the method of claim 28 and a pharmaceutically acceptable carrier.

32. A vector comprising, in operable linkage from 5' to 3':(a) a 5' long terminal repeat (LTR);(b) a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) protein, wherein the CAR protein comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain;(c) a nucleic acid sequence encoding a first peptide linker;(d) a nucleic acid sequence encoding a tag suitable for detection, selection, or depletion of a host cell expressing the CAR protein; and(e) a 3' long terminal repeat (LTR);33. The vector of claim 1, wherein vector is selected from the group consisting of retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors.

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